The goal of this research is to elucidate the mechanisms underlying how DNA repair proteins locate and repair damaged DNA. The health implications of this process extend to breast and ovarian cancers, as these cancers are associated with defects in this pathway. Improved understanding of homologous recombination and DNA repair will in turn allow better understanding of the development of human cancers. The focus of this proposal is to determine how the DNA repair proteins Rad51 and Rad54 interact with DNA and with other protein components of homologous recombination, and how DNA molecules are aligned during homologous recombination. This proposal hypothesizes that the presynaptic complex formed by Rad51, Rad54, and ssDNA searches dsDNA for regions of homology using ATP hydrolysis to drive translocation. In addition, it is hypothesized that ATP hydrolysis is a vehicle for the removal of nucleosomes and other DNA binding proteins from the dsDNA that would otherwise hinder access to homologous regions of dsDNA. The specific aims of this project are to (1) investigate the biochemical mechanism used by the presynaptic complex to probe dsDNA for homologous sequence and to (2) detail the interaction of the presynaptic complex with nucleosomes bound to the dsDNA. To test this hypothesis, reactions involving fluorescently labeled DNA molecules, Rad51, and Rad54 will be observed using total internal reflection fluorescent microscopy. This technique is able to directly visualize individual proteins interacting with individual molecules of DNA. DNA repair is an absolutely essential process to maintain cellular health. The proposed research is directly relevant to cancer biology, and the successful completion of the experiments contained in this research will contribute immensely to understanding of various types of cancers.